Evolvability describes an organism's capacity for producing descendants that are better adapted to a given environment. Chronic infections of bacteria can become dominated by mutators over time because the elevated mutation rates of these strains make them more evolvable. Little is currently known about how mutations affecting other cellular processes impact microbial evolvability and about how evolvability changes at each mutational step during the prolonged adaptation of a microorganism to a novel environment. The proposed project consists of three integrated studies that aim to identify mutations that promote microbial evolvability. First, mutation accumulation lines will be used to establish baseline expectations for the effects of fitness changes on the relative evolvability of Escherichia coli strains. Next, the histories of mutations in a twenty-year E. coli long-term evolution experiment will be reconstructed to search for examples of mutations that were ultimately successful because they increased evolvability relative to competing genotypes. Finally, functional genomics techniques will be used to screen libraries of E. coli gene deletion and overexpression strains for genetic changes that increase evolvability with respect to different environments. At each step in this process, marker divergence experiments will provide a consistent measure of evolvability in terms of the local fitness landscape of a test genotype. Prospective experimental designs will be compared and data will be interpreted throughout these studies using a common set of population genetics models and simulations. Currently, I am investigating relationships between genetic regulation and evolvability in E. coli, the experimental evolution of E. coli with reduced genomes, and using pretermination codon avoidance to predict gene essentiality. I am also extending the Avida artificial life platform to enable studies of the evolvability of self-replicating computer progams exhibiting complex genetic regulation. Relevance: Understanding what kinds of mutations are likely to affect the evolvability of microorganisms has implications for holding the progression of chronic infections and the responses of pathogen populations to new therapeutic pressures in check. Methods for engineering more evolvable domesticated microbes would also be useful for green chemistry, renewable bioenergy, and biotechnology applications related to medicine.